The present invention is related to stable liposome compositions. More particularly, the invention is directed to the physical stabilization of organic acid derivatives of alpha tocopherol bilayers comprising stabilizer, for use as a pharmaceutical composition.
The salt forms of organic acid derivatives of alpha tocopherol, such as tris (tris(hydroxymethyl)aminomethane)-tocopherol hemisuccinate (t-THS) are known to form closed multilamellar vesicles with high trapping efficiencies and captured volumes at physiological pH values (Janoff et al., U.S. patent application Set. No. 911,138, filed Sep. 24, 1986, entitled "Alpha Tocopherol-Based Vesicles", and incorporated herein by reference; and Lai et al., 1985, Biochemistry, 24:1646-1661). THS also demonstrates polymorphic (e.g. bilayer to hexagonal.sub.II) phase behavior dependent upon pH or the presence of divalent cartons. For example, while aqueous suspensions of THS are in the bilayer phase at physiological pH and above (about pH 6.0-9.0), lowering the pH below about 6.0 or introducing divalent cartons such as Mg.sup.++ or Ca.sup.++ produces vesicle aggregation and fusion, and induces hexagonal phase (hex.sub.II) formation.
Neither the tocopherol acid succinate (hemisuccinate) nor the tris salt form of THS form or remain liposomes at acidic pH, this due to the formation of hex.sub.II structures at low pH. Alpha-THS is a branched single chain lipid that differs dramatically in structure from other lipids known to exhibit bilayer to hexagonal phase polymorphism, such lipids being for example, phosphatidylethanolamine, cardiolipin, and mono-diacyl glycerides.
In previous studies using alpha tocopherol hemisuccinate (THS) in liposomal drug delivery systems, the bioactive agent to be entrapped or associated with the THS was stable at physiological pH values. In such drug delivery systems, the requirements for physiological pH for bilayer state of THS was met by the similar pH requirements for the drug. However, certain drugs, for example pilocarpine, are catalytically active in such unprotonated form as at physiological pH, and therefore require delivery systems of more acidic pH. Such a system heretofore was not compatible with THS as the delivery system, due to the pH requirements of THS in the bilayer state, since such conditions make THS highly susceptible to hydrolysis and therefore degradation. Moreover, pilocarpine itself is unstable at high pH, where it undergoes base-catalyzed hydrolysis. At lower pH, pilocarpine is protonated and more stable (Chung et al., 1970, J. Pharm. Sci., 59(9):1300-1305). Pilocarpine among other drugs thus requires, and is therefore preferably associated with bilayer systems at lower (acidic) pH.
Thus, in lipid-based delivery systems employing THS and drugs such as pilocarpine, the requirement for acidic conditions of the drug appears incompatible with the requirement for the bilayer form (neutral to basic conditions) for the THS. Due to the polymorphic phase behavior of the THS molecule, which produces the above-mentioned hex.sub.II phase and precipitation in solution at acidic pH, and the requirement for acidic pH of the drug, we have developed a new system utilizing stabilizers that maintain the bilayer configuration of the THS at both acidic and alkaline pH. Under these conditions, where stabilizer is present, the THS is stabilized in solution at acidic pH. This system utilizes THS liposomes which associate with or entrap bioactive agent which tolerate or require low pH conditions, for example, drugs having imidazole groups, where the final pH of the preparation is acidic.
Stabilizers are defined as compounds which allow the formation and/or maintenance of the bilayer state of d-alpha-tocopherol acid succinate based liposomes in an acidic pH environment. Materials which have been found to perform that function generally belong to the classes of compounds commonly known as surfactants and detergents. In the present invention, stabilizers (detergents) such as the pharmaceutically acceptable excipients polyoxyethylene alkyl ethers (such as polyoxyethylene-4-laurel ether, polyoxyethylene-23-laurel ether, and a combination thereof, known also by the respective names Laureth-4, Laureth-23, and Laureth-12, ICI Americas, Inc.); the appended numbers denoting the average number of ethylene oxide units in the polyethylene glycol segment), were found to stabilize THS into the bilayer phase at acidic pH.
We have surprisingly discovered a system using THS-stabilizer bilayers which may be adjusted to acidic pH, thereby accomodating drugs that require low pH conditions. In this system, a THS-stabilizer bilayer is formed at low pH, for example at acidic pH, for example at between about pH 2.0 and 6.5, more preferably about pH 4.0-5.0, most preferably about pH 4.5. A drug requiring such low pH conditions may then be added to the preparation; alternatively, the drug may be added prior to adjustment of the pH. We have further discovered that when this preparation was adjusted to neutral pH (e.g., physiological pH), the bilayer phase of the system is preserved, and the viscosity of the solution may increase. Such pH adjustment may effectively take place upon addition of the solution to body fluids, e.g. in the case of ocular pilocarpine and the tear film. For example, addition of the acidic liposomal THS pilocarpine solution to the eye results in an increase in viscosity which may increase the time the pilocarpine is in contact with the eye tissues. An increase in contact time is known to be directly related to the enhanced uptake of drugs by the eye tissues (Chrai et al., 1974, J. Pharm, Sci., 63:1218, Lee at al., 1974, J. Pharm. Sci., 63:721).
The mode of delivery of the bioactive agent of the present invention is preferably via association of the agent with lipid, (e.g., THS), via entrapment in the liposome, association with the outside of the liposome, or inside the lipid bilayer. Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single membrane bilayer) or multilamellar vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient towards the center of the bilayer while the hydrophilic "heads" orient towards the aqueous phase.
The original liposome preparation of Bangham et al. (J. Mol. Biol., 1965, 12:238-252) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell," and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles described by Papahadjopoulos et al. (Biochim. Biophys. Acta., 1968, 135:624-638), and for large unilamellar vesicles.
As mentioned above, a variety of sterols and their water soluble derivatives have been used to form liposomes; see specifically Janoff et al., PCT Publication No. 85/04578, Oct. 24, 1985, entitled, "Steroidal Liposomes." Mayhew et al., PCT Publication No. 85/00968, Mar. 14, 1985, described a method for reducing the toxicity of drugs by encapsulating them in liposomes comprising alpha-tocopherol and certain derivatives thereof. In a liposome-drug delivery system, a bioactive agent such as a drug is entrapped in or associated with the liposome and then administered to the patient to be treated. For example, see Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871; Schnieder, U.S. Pat. No. 4,114,179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.
The present invention solves the problem of delivery of an acid requiring or acid tolerating drug at acidic pH, with THS. By combining THS with stabilizer at an acidic pH, bilayer conditions of THS are preserved. Further, following the adjustment of the pH to neutral, the system increases in viscosity, enhancing the contact time between drug and body tissue. The system may also comprise a drug, such as an acid requiring or acid tolerating drug, for example, pilocarpine.
In the present invention, liposomes comprising the THS, bioactive agent, and stabilizer are adjusted to acidic pH and administered to a subject.